Piloting the administration of Sorafenib 100 mg/kg/d via oral gavage and measuring blood pressure by the tail-cuff method

Procedural outline

Turn on machines / heaters. Put mice in tailcuff room and let the room and mice acclimate to appropriate temperature for ~30-60 mins. Then, check cuffs for leaks. Put mice into restraints and perform 5 acclimation cycles + 20 recorded cycles.

When placing mice into restraints,
1. Quickly place nose cuff on to avoid letting them turn around in the restraint
2. Make sure that you can see them breathing…
3. Maximize tightness of fit and breathing

Metadata Analysis

This workbook is set up to analyze two groups of mice! Just run and enjoy (you’ll probably need to change out drug names…)

Metadata

group	Specimen Name	Mouse Unique ID	Gender	Notch	DOB	Wean Date	CageID	Current Age (weeks)	Date ready for Telemetry implant	New CageID	Status	Date of death	Machine ID	Average body weight (g)
vehicle	M1	1A	Male	NN	2021-03-25	2021-04-20	662055	9.285714	2021-05-20	NA	Alive	NA	1	26.4
sorafenib	M2	2A	Male	RN	2021-03-25	2021-04-20	662055	9.285714	2021-05-20	NA	Alive	NA	1	24.9
sorafenib	M3	3A	Male	LN	2021-03-25	2021-04-20	662055	9.285714	2021-05-20	NA	Alive	NA	1	26.2
vehicle	M4	4A	Male	BN	2021-03-25	2021-04-20	662055	9.285714	2021-05-20	NA	Alive	NA	1	25.7
vehicle	M5	5A	Male	DR	2021-03-25	2021-04-20	662055	9.285714	2021-05-20	NA	Alive	NA	1	27.5
sorafenib	M6	1B	Male	NN	2021-03-30	2021-04-13	662047	8.571429	2021-05-25	NA	Alive	NA	2	25.1
vehicle	M7	2B	Male	RN	2021-03-30	2021-04-13	662047	8.571429	2021-05-25	NA	Alive	NA	2	25.7
vehicle	M8	3B	Male	LN	2021-03-30	2021-04-13	662047	8.571429	2021-05-25	NA	Alive	NA	2	24.9
sorafenib	M9	4B	Male	BN	2021-03-30	2021-04-13	662047	8.571429	2021-05-25	NA	Alive	NA	2	23.4
sorafenib	M10	5B	Male	DR	2021-03-30	2021-04-13	662047	8.571429	2021-05-25	NA	Alive	NA	2	25.3

Inspect accepted cycles and changes in mouse body weight over time

Average animal body weight

Joining, by = “Specimen Name”

Animal body weight change over time

Joining, by = “Specimen Name”

Blood Pressure Data Analysis

Filtering out days that had less than ‘x’ cycles

Removed days/Specimens with less than 5 cycles:

Specimen Name	Date	group	# cycles	reason
M3	2021-05-25	sorafenib	1	Low cycle count
M7	2021-05-25	vehicle	4	Low cycle count

Removing outliers

Detect outliers BP measurements using boxplot methods. Boxplots are a popular and an easy method for identifying outliers. There are two categories of outlier: (1) outliers and (2) extreme points. Values above Q3 + 2xIQR or below Q1 - 2xIQR are considered as outliers. Q1 and Q3 are the first and third quartile, respectively. IQR is the interquartile range (IQR = Q3 - Q1). This method is more robust than STDEV based outlier detection because outliers can skew the mean and STDEV of a sample.

Here, outliers are nominated based on daily blood pressure recordings, so as to not throw out data on treatment days when the blood pressure is expected to rise above the average.

Additionally, we remove mice that are too ‘volatile’ after trianing period has finished.

Plot the data over time and visualize the variance per day, per sample with boxplots, over all days

Pilot 2 | sorafenib 100 mg/kg/d

Dates

Phase	first	last	date_range	Number of Days
training	2021-05-25	2021-05-28	2021-05-25 to 2021-05-28	4
vehicle	2021-05-29	2021-05-29	2021-05-29 to 2021-05-29	1

Assess BP of randomly assigned groups before getting treatment

3-day rolling average

Take the last 3-days of an interval and average them for more realistic averages

summarise() has grouped output by ‘Specimen Name’, ‘group’. You can override using the .groups argument. summarise() has grouped output by ‘group’. You can override using the .groups argument.

Time-series data

For completeness, here is the time series data of each mouse across each Phase of the experiment:

Time-series diff by individual mouse from vehicle average to end of treatment

Time-series diff by group

These are the average blood pressures of each mice across each Phase of the experiment

Booklet usage instructions

Download excel data after finishing the experiments onto thumb drive

Copy the data into a master excel file with two sheets, making sure that there’s only one header (at the very top of the page).

1. You’ll need to add in two columns manually to this master sheet: Date and Phase. Phase can take one of 4 values: “training”, “baseline”, “vehicle”, “treatment”. Training data ultimately gets removed, but included in the data sheet for completeness. First sheet should look like this:

2. Second sheet should be created manually based on your mice. Fill in the various fields.